1. Field of the Invention
This invention relates to a growth medium derived from human brain endothelial cells (HUBEC) and the methods of utilizing said growth medium to expand bone marrow stem cells.
2. Description of the Prior Art
The development of an ex-vivo system which supports the proliferation and expansion of the most primitive hematopoietic stem cells (HSC) would have direct application to the fields of gene therapy and stem cell transplantation. Identification and characterization of the optimal culture conditions for the expansion of long-term repopulating cells is a requirement for gene therapy protocols and other stem cell-based therapies.
Various cytokine combinations and liquid culture methods have been shown to support the proliferation of CD34+ HPC in vitro, but the most primitive CD34+CD38xe2x88x92 cells are frequently lost due to differentiation and cell death [1-6]. In contrast, other investigations have demonstrated that when human HPC are co-cultured in contact with autologous, allogeneic, and xenogeneic bone marrow stroma, a small percentage of long term culture initiating cells (LTC-IC) can be maintained over several weeks [7-9]. Similarly, others have reported the expansion and differentiation of LTC-IC and CFC in stroma-free liquid suspension cultures using exogenous cytokines plus conditioned medium from bone marrow stromal cultures [10-12]. Most recently, it was reported that human cord blood CD34+ cells could be maintained in stroma-free liquid cultures in the presence of flt-3 ligand, megakaryocyte growth and development factor (MGDF), SCF, and IL-6 for up to 10 weeks without losing their ability to repopulate NOD/SCID mice [13].
Vascular endothelium, reticuloendothelial elements, and hematopoietic cells of all types have been postulated to arise from hemangioblasts, a primitive embryonic cell of mesodermal origin [14,15]. During the earliest stages (day 7-8 postcoitum) of mammalian embryonic hematopoiesis, primitive hematopoietic stem cells are found encased in blood islands which derive from aggregates of mesodermal cells which have colonized the embryonic yolk sac [16]. Bone marrow, umbilical vein, and murine yolk sacendothelial cell lines have been shown to elaborate a number of growth factors that regulate early hematopoiesis [17-20]. In addition, the long term proliferation and differentiation of myeloid, erythroid, and megakaryocytic progenitor cells has been demonstrated in vitro using microvascular endothelial cells derived from adult bone marrow and embryonic yolk sac [18,19]. However, the fate of the most primitive CD34+CD38xe2x88x92 progenitor cells following co-culture with endothelial cell monolayers has not been well demonstrated. Previously, we reported that a primary porcine microvascular endothelial cell line (PMVEC) supports a rapid and robust expansion of human hematopoietic cells exhibiting the primitive CD34+CD38xe2x88x92 phenotype [21,22]. Unlike other reported co-culture systems, we have demonstrated that CD34+CD38xe2x88x92 cells expanded on brain endothelium retain the ability to successfully engraft in vivo in both a SCID-Hu bone model [23] and in lethally irradiated baboons [24].
Human brain vascular endothelial cells are similar to other sources of endothelial cells in that they develop cobblestone morphology in-vitro [25], and they express cell adhesion.molecules (selectins, integrins) which mediate the xe2x80x9crollingxe2x80x9d, adherence, and trafficking of leukocytes [26,27]. Based upon our observations of the hematopoietic capacity of PMVEC and recognizing the limitations of applying a porcine endothelial cell line in human clinical studies, we isolated. primary human brain endothelial cells (HUBEC) and evaluated their capacity to support the ex-vivo expansion of human CD34+CD38xe2x88x92 cells. Our results indicate that human brain endothelial cells support a unique expansion and apparent self-renewal of the most primitive CD34+CD38xe2x88x92 HPC at a level comparable to our observations with porcine endothelial cells. Further investigations evaluating the in vivo repopulating potential of HUBEC-expanded HPC will be important in implementing future gene therapy, cord blood expansion, and stem cell transplant protocols.
Accordingly, an object of this invention is a growth medium based on human brain endothelial cells (HUBEC).
Another object of the invention is the growth factor contained within the medium that is elaborated by the HUBEC and promotes the expansion of primitive CD34+CD38xe2x88x92 bone marrow stem cells.
A further object of this invention is a method for expanding the population of primitive CD34+ CD38xe2x88x92 bone marrow stem cells.
Yet another object of this invention is the treated, concentrated product of the growth medium containing the growth factor.
An additional object of the invention is a growth medium that can be used for GMP production of expanded cells.
These and additional objects of the invention are accomplished by human brain endothelial cells (HUBEC) that can serve as a uniquely supportive hematopoietic microenvironmnent.